Entorhinal Neurons Exhibit Cue Locking in Rodent VR

Abstract

The regular firing pattern exhibited by medial entorhinal (mEC) grid cells of locomoting rodents is hypothesized to provide spatial metric information relevant for navigation. The development of virtual reality (VR) for head-fixed mice confers a number of experimental advantages and has become increasingly popular as a method for investigating spatially-selective cells. Recent experiments using 1D VR linear tracks have shown that some mEC cells have multiple fields in virtual space, analogous to grid cells on real linear tracks. We recorded from the mEC as mice traversed virtual tracks featuring regularly spaced repetitive cues and identified a population of cells with multiple firing fields, resembling the regular firing of grid cells. However, further analyses indicated that many of these were not, in fact, grid cells because: (1) when recorded in the open field they did not display discrete firing fields with six-fold symmetry; and (2) in different VR environments their firing fields were found to match the spatial frequency of repetitive environmental cues. In contrast, cells identified as grid cells based on their open field firing patterns did not exhibit cue locking. In light of these results we highlight the importance of controlling the periodicity of the visual cues in VR and the necessity of identifying grid cells from real open field environments in order to correctly characterize spatially modulated neurons in VR experiments.

Keywords: entorhinal cortex; grid cell; path integration; spatial cognition; virtual navigation; virtual reality.

Figure 1

Spatially periodic activity in real world (RW) and virtual reality (VR) environments, a subset of non-grid medial entorhinal (mEC) cells show cue-locking in VR environments. (A) Spatial activity from two example grid cells—identified based on RW recordings (right)—in each of the three VR environment (left). Repeating cues are indicated as colored bars in the background of the VR plots. Cells are color coded such that the title on RW ratemaps matches line color on the VR plots. (B) Similar to (A), spatial activity from two cells which were (incorrectly) classified as grids cells based on VR activity (left) but not based on RW open field activity (right). Despite the regularity of their firing patterns in VR, these cells showed no clear grid-like firing in RW and only limited spatial responses. (C) Cue-locking in grid cells (n = 15, identified from RW) was investigated using spatial auto-correlograms (SACs). Plots show mean (black line) ± SEM (light green shade area) SACs across cells (left y-axis). Note the lack of periodicity corresponding to the frequency of cues in the VR environments (indicated by gray and orange bands). The overlaid color-coded dots represent the dominant spatial frequency in the 20–180 cm range detected from the SAC of each grid cell—the distribution of these points is indicated by the red line (right y-axis). (D) Proportion of grid cells exhibiting cue-locking (yellow) and no cue-locking (blue) in each VR environment (left) and proportion showing cue-locking in multiple VR environments (right).

Figure 2

A sub-population of non-grid cells exhibit pronounced spatial periodicity in 1D VR environments. Examples of co-recorded non-grid periodic cells from two animals (A, 10 cells from mouse m3504: B, five cells from mouse 3193) shown as ratemaps in both VR (top row) and RW (bottom rows). In VR, the firing rate of cells is plotted against position in each environment (A–C) with the periodicity of repeating cues indicated in the background (gray and orange). In RW the ratemaps of the same color-coded cells in VR are shown together with mean and peak firing rate, grid score, stability, and spatial information. Despite the regularity of the firing pattern patterns in VR, these cells neither showed clear grid-like firing pattern nor spatial firing of any kind in RW.

Figure 3

Spatial frequency of the periodic non-grid cells. Within VR environments, periodic non-grid cells exhibited regular firing at the same spatial frequency as the underlying repetitive visual cues, unlike grid cells which showed weaker spatial periodicity of varying frequencies. (A) SAC of all non-grid periodic cells across VR environments. Note the clear peaks centered on the spatial frequency of the repeating cues of each environment (gray block for Environment A and B and gray and orange in Environment C). (B) Mean (black line) ± SEM (light green shaded area) of the SAC (left y-axis) across cells within each environment showing clear coincidence with the frequency of the repetitive cues (gray block). Colored dots indicate the dominant spatial frequency of each cell (color matches lines in A) and were used to compute the kernel density estimate (red line, right y-axis). (C) Histograms showing (left) percentages of non-cue locked (blue) and cue-locked (yellow) periodic cells within each VR environment. Right, Histograms showing percentages of periodic cells exhibiting cue locking in multiple environments. Most cells (>85%) displayed cue-locking in at least one VR environments.

Figure 4

Non-grid periodic cells were strongly modulated by the frequency of the repetitive segments in each VR but were not clustered at specific phases within each segment. (A) Rate maps for each color-coded cell across VR environments showing mean firing rate as function of location within the repeating segment. Note the differences in the peak firing rate and location of the spatial tuning curves across cells. (B) Ratemaps of all cue-locked cells sorted according to location of peak firing. For comparison across environments location within each repeated segment has been converted to a phase (radians). Note the sequence of firing within each environment with no strong preference for any particular phase. (C) Ratemaps from environments A (Left) and B* (Middle) sorted according to the order of their peaks in A. (Right) Cross-correlation between A and B* shows a significant peak (vs. 1,000 shuffles, purple line also indicates shuffle confidence interval), suggesting a tendency for the relative phase of ratemaps to be preserved between environment A and B.